Aiouea padiformis extract exhibits anti-inflammatory effects by inhibiting the ATPase activity of NLRP3

Inflammation is implicated as a cause in many diseases. Most of the anti-inflammatory agents in use are synthetic and there is an unmet need for natural substance-derived anti-inflammatory agents with minimal side effects. Aiouea padiformis belongs to the Lauraceae family and is primarily found in tropical regions. While some members of the Aiouea genus are known to possess anti-inflammatory properties, the anti-inflammatory properties of Aiouea padiformis extract (AP) have not been investigated. In this study, we aimed to examine the anti-inflammatory function of AP through the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and elucidate the underlying mechanisms. Treatment with AP inhibited the secretion of interleukin-1 beta (IL-1β) mediated by NLRP3 inflammasome in J774A.1 and THP-1 cells without affecting the viability. In addition, AP treatment did not influence NF-κB signaling, potassium efflux, or intracellular reactive oxygen species (ROS) production—all of which are associated with NLRP3 inflammasome activation. However, intriguingly, AP treatment significantly reduced the ATPase activity of NLRP3, leading to the inhibition of ASC oligomerization and speck formation. Consistent with cellular experiments, the anti-inflammatory property of AP in vivo was also evaluated using an LPS-induced inflammation model in zebrafish, demonstrating that AP hinders NLRP3 inflammasome activation.


AP specifically reduces NLRP3 inflammasome activation
We screened extracts from 45 different plant species native to Nicaragua for NLRP3 inhibition activity.The reduction in IL-1β secretion was assessed by immunoblotting and the relative expression was quantified using ImageJ (ver.1.54d) (Supplemental Table 1).Among the 45 plant extracts tested, we selected candidates that did not affect cell viability, showed concentration-dependent reduction in IL-1β secretion, and did not inhibit the NF-κB signal.Among these selected candidates, AP appeared to effectively reduce IL-1β and was therefore used for subsequent experiments.
To confirm the anti-inflammatory effects of AP, we first measured the viability of mouse J774A.1 and human THP-1 cells after AP treatment.The viability of both types of cells was not altered even when treated with the highest concentration of 100 μg/mL of AP (Fig. 1a, b).We then investigated the effect of AP treatment on IL-1β secretion mediated by NLRP3 inflammasome.When LPS-primed J774A.1 cells were exposed to nigericin or adenosine triphosphate (ATP), well-known NLRP3 activators, they showed a significant increase in IL-1β secretion.However, when the cells were treated with both the activators and AP, IL-1β and IL-18 secretion decreased in a concentration-dependent manner (Fig. 1c, d, Supplemental Fig. 1).The effects of AP were compared with those of MCC950, an NLRP3-specific inhibitor 39 .As shown in Fig. 1c, d, a high concentration of AP inhibited NLRP3 activation to a similar extent as MCC950.Similar results were obtained when phorbol 12-myristate 13-acetate www.nature.com/scientificreports/(PMA)-primed THP-1 cells were treated with nigericin alone or in combination with AP or MCC950 (Fig. 1e).Lastly, to confirm whether AP selectively inhibits NLRP3 or also affects other inflammasomes, we activated Absent in Melanoma 2 (AIM2) and NOD-like receptor family CARD domain-containing protein 4 (NLRC4) inflammasomes by treating the cells with dsDNA or flagellin, respectively, followed by AP treatment.When we measured the secretion of IL-1β, AP treatment did not affect IL-1β production, indicating that AP specifically blocked the activation of the NLRP3 inflammasome (Fig. 1f, g).

AP does not impact the NF-κB signaling pathway
We then investigated how AP suppresses the NLRP3 inflammasome.The production of IL-1β involves two steps: the priming phase, activating NF-κB to increase pro-IL-1β transcription, and the activation phase, inducing inflammasome formation.To check if AP reduces NF-κB activity during the priming step, we assessed NF-κB activity by an NF-κB luciferase reporter gene assay.Treatment with tumor necrosis factor-alpha (TNF-α) significantly increased NF-κB signaling, and co-treatment with TNF-α and AP had no effect on the elevated NF-κB signaling (Fig. 2a).To further confirm these findings, we analyzed phospho p65 levels, an NF-κB activity indicator.AP treatment did not affect the levels of phosphorylated p65 (Fig. 2b).These results indicate that AP suppresses the NLRP3 inflammasome without affecting NF-κB signaling.

AP suppression of the NLRP3 inflammasome is not dependent on K + efflux, increased intracellular ROS level, or mitochondrial membrane potential
As AP did not have any influence on the priming step, we investigated its potential impact on the activation step.In the process of activating the NLRP3 inflammasome, two key events are the efflux of potassium ions or an increase in intracellular ROS.Blocking these events can suppress NLRP3 inflammasome activity.To explore if AP could hinder NLRP3 activation by affecting potassium efflux, we treated cells with imiquimod, which activates the NLRP3 inflammasome independently of K + efflux 40 .As shown in Fig. 3a, AP suppressed NLRP3 activation by imiquimod in a concentration-dependent manner, suggesting that the inhibition of the NLRP3 inflammasome by AP is not dependent on K + efflux.To determine whether AP regulates intracellular ROS levels  www.nature.com/scientificreports/ to inhibit the NLRP3 inflammasome, we measured ROS levels after AP treatment.While ATP treatment increased ROS levels, AP did not influence intracellular ROS level.However, when cells were treated with N-acetylcysteine (NAC), a well-known ROS scavenger, ROS levels significantly decreased (Fig. 3b).NLRP3 activators can induce mitochondrial instability, and the resulting mitochondrial dysfunction may lead to the activation of the NLRP3 inflammasome.Therefore, we examined alterations in mitochondrial function by measuring the mitochondrial membrane potential in cells treated with ATP, followed by AP or not.The decrease in the ratio of red to green fluorescence intensity indicates mitochondrial depolarization.While mitochondrial membrane potential exhibited a significant alteration with ATP treatment, AP treatment did not mitigate the change in membrane potential (Fig. 3c).Therefore, we found that K + efflux, intracellular ROS, and mitochondria are not implicated in the mechanism by which AP inhibits the activation of the NLRP3 inflammasome.

AP inhibits ATPase activity and ASC oligomerization of the NLRP3 inflammasome
Never in Mitosis Gene A-related kinase 7 (NEK7) is essential for promoting oligomerization of the NLRP3 inflammasome, which is necessary for its activation 41,42 .We examined the effect of AP on the interaction between NEK7 and the NLRP3 using co-immunoprecipitation experiment.No significant differences in the amount of NEK7-NLRP3 complexes were observed between the untreated samples and those treated with AP.Therefore, we confirmed that AP did not interfere with the interaction of NEK7 and NLRP3 (Fig. 4a).ATPase activity is essential for the assembly of the NLRP3 inflammasome 43 .Many NLRP3 inhibitors are known to impede the ATPase activity of NLRP3 44,45 ; therefore, we set out to determine whether AP affects ATPase activity.As shown in Fig. 4b, AP significantly reduced the ATPase activity of NLRP3, and the extent of reduction was nearly identical to the well-known ATPase activity inhibition by MCC950.We subsequently investigated ASC oligomerization, a marker of inflammasome assembly 46 .Since the ATPase activity of NLRP3 is crucial for inflammasome oligomerization, it can be predicted that the oligomerization of ASC, a common component of the inflammasome, will also be inhibited by AP.As expected, AP treatment attenuated ASC oligomerization in a concentration-dependent manner (Fig. 4c).These observations were further confirmed by assessing the extent of ASC speck formation using confocal microscopy, which represents inflammasome assembly.Consistent with previous data, speck formation was also significantly reduced by AP (Fig. 4d, e).These results revealed that the APmediated inhibition of NLRP3's ATPase activity can be attributed to the prevention of inflammasome assembly.
To identify the major bioactive component in AP responsible for the IL-1β inhibitory activity, AP was subjected to MPLC.Supplemental Fig. 2a shows the identification of the main chromatography of AP.Among the AP fractions (AP1-AP11), the AP7 fraction was found to contain the most abundant components (Supplemental Fig. 2b).Consequently, the AP7 fraction was subjected to recycle-HPLC, resulting in the isolation of five fractions (AP71-AP75).Among them, Compound 1 (3.1 mg, tR = 54.1 min) was isolated from subfraction AP75 using preparative HPLC (acetonitrile-water, 15:85 to 85:15 in 90 min).The structure of the isolated compound was elucidated by MS and 1D/2D NMR data analyses and was confirmed to be diethyl phthalate upon compared with the corresponding data reported in the literature (Supplemental Fig. 2a).

AP alleviates inflammatory responses induced in the zebrafish embryos
To evaluate the anti-inflammatory effects of AP at the organism level, we used a zebrafish (Danio rerio) model.Zebrafish embryos are an effective tool to study inflammation during early development 47,48 .Inflammation can be induced through various methods, including LPS immersion, direct injection of LPS into the yolk sac, and tail-cut injuries 36 .
We conducted a primary experiment by inducing inflammation in 1 day post-fertilization (dpf) zebrafish embryos by treating the larvae with 10 µg/mL LPS.Following an induction period of 2 days, the embryos were fixed.The resulting inflammatory response was assessed by monitoring the number of myeloid cells using Sudan Black B staining 49 .Inflammation was successfully induced in the LPS-treated zebrafish, and co-treatment with 10 µg/mL of AP markedly reduced this inflammation (Fig. 5a).Further quantification revealed a significant reduction in the levels of the inflammatory markers in embryos treated with AP compared to those that were not (Fig. 5b).Additionally, under the same conditions, whole-mount hybridization (WISH) analysis targeting neutrophil-specific mpx expression revealed that AP treatment reduced the LPS-induced increase in mpx-positive cells (Fig. 5c).In addition to the LPS-immersion experiments, we induced inflammation by directly injecting LPS into the zebrafish yolk.This approach led to noticeable inflammation, particularly around the injection site and the anterior part of the embryos.The enhanced inflammatory response was significantly alleviated by incubation with AP (Fig. 5d, e).
Taken together, the findings of our experiments on the zebrafish model verify the potent anti-inflammatory effects of AP at the organismal level, further emphasizing the broader potential of this natural product as an anti-inflammatory agent.

Discussion
Elevated IL-1β expression is involved in many diseases, and the severity of these diseases is often correlated with the magnitude of IL-1β upregulation 1 .Consequently, strategies aimed at blocking IL-1β have emerged as promising therapeutic approaches for inflammatory diseases.IL-1β secretion is intricately controlled by various inflammasomes, requiring research to understand the regulation of these inflammasomes and for the development of inhibitors.While most inflammasomes directly recognize DAMPs or PAMPs, NLRP3 detects cellular homeostatic changes, without direct recognition of specific components, thereby contributing to the pathogenesis of various diseases where such alterations are observed, and also creating a feedback loop that exacerbates disease symptoms.Given the association of NLRP3 with numerous diseases, ongoing research is actively exploring technologies specifically designed to inhibit NLRP3.While ongoing research actively explores small molecules directly inhibiting NLRP3, none of these compounds has yet received FDA approval due to concerns about their toxicity.To address these safety concerns, extensive efforts are being made to investigate natural products as potential alternative NLRP3 inhibitors.
Therefore, we examined the anti-inflammatory properties of about 200 different plant extracts from Costa Rica and Nicaragua which had not been studied before.Among the investigated extracts, we identified potent NLRP3 inhibitory effects in Guarea 50 and Trichospira from Costa Rica, and these findings have been recently submitted for publication.Given that among the 45 Nicaraguan compounds tested, AP exhibited the highest level of anti-inflammatory activity, and considering the numerous studies reporting anti-inflammatory effects in plants belonging to the same genus, we believe it is worthwhile to investigate the anti-inflammatory function of AP.In this study, we aimed to demonstrate the inhibitory effect of AP on NLRP3 inflammasome activation and elucidate its underlying mechanism.As recently developed NLRP3 inhibitors, such as MCC950, exert their mechanism through the inhibition of NLRP3's ATPase activity 39 , thereby disrupting the assembly of the NLRP3 inflammasome and many NLRP3 inhibitors currently under development also target the ATPase activity of NLRP3, we also investigated whether AP could similarly inhibit the ATPase activity of NLRP3, leading to the decreased ASC oligomerization and speck formation.As expected, AP effectively suppressed the ATPase activity (Fig. 4c-e).Fundamentally, to inhibit ATPase activity, it is necessary to directly bind to NLRP3.Therefore, we analyzed the composition of the extracts using HPLC to identify which components of AP bind to NLRP3.The HPLC analysis of AP revealed that diethyl phthalate is the most abundant component in AP.Diethyl phthalate is commonly used in the manufacturing of plastics, insecticides, cosmetics, and aspirin.In addition, since it often used as plasticizers for polyvinyl, polyvinyl chloride, or cellulose resins, there have been reports on the potential risks associated with long-term exposure to it [51][52][53][54][55] .While there are studies suggesting that diethyl phthalate extracted from plants might be artificial due to contamination in various containers or during the extraction process 56,57 , many studies have indicated its extraction from bacteria and plants, highlighting its anti-inflammatory properties 58,59 .However, in this study, as we could not confirm whether diethyl phthalate can modulate the ATPase activity of NLRP3, it is essential to investigate whether various substances collaborate to inhibit NLRP3 or if they can individually inhibit NLRP3.Additionally, the impact of each isolated compound on cells should be determined.In addition, it is important to identify the substances among the discovered active compounds that directly bind to NLRP3, inhibiting its ATPase activity.Future research involving extensive collection of AP could allow for the analysis of additional substances within AP.This could lead to the discovery of new compounds directly binding to NLRP3 or investigating various activities of diethyl phthalate.
Our study suggests the potential of AP as an NLRP3 inhibitor; however, many questions remain.First, as the composition of AP may be influenced by environmental factors, the separation and purification of bio active substances are challenging due to the time required for large-scale collection under the same conditions.However, securing the confirmed active compounds is expected to be a crucial starting point in the search for NLRP3 inhibitors from plant extracts.Second, in our experiment, AP significantly reduced the ATPase activity of NLRP3 and ASC speck formation but did not regulate its binding with NEK7 (Fig. 4).However, the association of NEK7 with NLRP3 is the essential for the assembly of the NLRP3 inflammasome, including ASC speck formation 24 .Recently, several studies have suggested that the phosphorylation of NLRP3 is crucial for the activation of the NLRP3 inflammasome [60][61][62] .In addition, recent studies have reported that NLRP3 exists in an inactive CAGE form 63 .Therefore, additional research is essential to verify whether AP influences phosphorylation or preserves the CAGE model, thereby impeding activation.Third, as gain-of-function mutations in NLRP3 gene like R260W, D303G, and E311K cause autoinflammatory disease, CAPS, we need to assess the potential utilization of newly discovered substances as a treatment for CAPS patients by determining where they bind on NLRP3.In the case of the well-known NLRP3 inhibitor MCC950, it had no effect on CAPS patients with specific mutations 64 .Therefore, it is crucial to identify the binding location of the substances we have discovered on NLRP3 and understand how they function in CAPS patients.Finally, the medicinal use of Aiouea species is well-known, and some species have been analyzed for their constituent components.However, there is currently a lack of substantial scientific evidence regarding their potential medicinal properties, especially in terms of demonstrating strong anti-inflammatory activity.To determine whether specific medicinal uses are associated with Aiouea species, it is necessary to explore detailed research or traditional practices specific to each species.

Conclusion
These results show that AP suppresses ATP hydrolysis through the NACHT domain of NLRP3, which possesses ATPase activity, thereby blocking conformational changes in NLRP3.Our results suggest that compounds in the AP may bind directly to the NACHT domain of NLRP3, thus inhibiting its ATPase activity.Overall, this study could provide insight into the anti-inflammatory properties of AP and potentially lead to the development of novel anti-inflammatory therapies for disorders involving NLRP3 inflammasome activation.

Plant sample collection
The collection and preservation of plants material were conducted under the agreement for sustainable exploitation of the Nicaraguan flora between International Biological Material Research Center (IBMRC) in Korea Research Institute of Bioscience and Biotechnology (KRIBB) of Korea and Universidad Nacional Autonoma de Nicaragua-Leon of Nicaragua.All research samples follow the regulation of Nicaragua institutional, national, and international guidelines and legislation, with all necessary permissions and licenses obtained to ensure compliance with ethical and legal requirements.

Measurement of cellular ROS assay
In a 4-well culture slide, J774A.1 cells (2 × 10 5 cells/well) were seeded 1 day before the experiment.Cells were primed by treating them with LPS (100 ng/mL) for 5 h, followed by AP (50 μg/mL) treatment for 2 h.The medium was removed, and the cells were washed with 1× buffer (250 μL/well).Subsequently, we treated the cells with DCFDA solution (20 μM) for 1 h at 37 ℃ in the dark.The solution was removed, and cells were washed with 1× buffer (250 μL/well), and activated with ATP (5 mM) for 5 min.The cells were mounted in a medium containing DAPI for nuclear staining.Images were obtained using a confocal laser-scanning microscope (Carl Zeiss, LSM710).

Measurement of mitochondrial membrane potential
J774A.1 cells (2 × 10 5 cells/well) seeded in a 4-well culture slide primed with LPS (100 ng/mL) for 3 h, followed by treatment with AP (50 μg/mL) for 2 h.For measuring mitochondrial membrane potential, we washed the cells with 1× phosphate-buffered saline (PBS) and stained with JC-1 dye (10 μM) for 10 min at 37 ℃ in the dark.After staining, the cells were activated with ATP (5 mM) for 5 min.Images were obtained using a confocal laserscanning microscope (Carl Zeiss, LSM710).

ASC oligomerization and ASC speck staining
To investigate ASC oligomerization, pellets of THP-1 cells (1.5 × 10 6 cells/well in 12 well plate) were resuspended with PBS for washing and incubated with DSS (2.5 mM) for 30 min at 25 ℃.ASC oligomerization was analyzed by immunoblotting.

Figure 2 .
Figure 2. AP does not affect the NF-κB signaling pathway.(a) 293 T cells were transfected with the pGL4.32 luciferase reporter vector and treated with TNF-α (20 ng/mL) for 5 h with or without AP.Luciferase activity was analyzed using the Promega Bright-Glo™ Luciferase Assay System.(b) LPS-primed J774A.1 cells were treated with AP for 2 h and activated for 30 min with nigericin (10 μM).IL-1β in the supernatants (Sup), phospho-NF-κB and NF-κB in the soluble lysates (Lys) were analyzed using a Western blot.

Figure 4 .
Figure 4. AP blocks NLRP3 inflammasome assembly by hindering ATPase activity of NLRP3.(a) HEK 293FT cells were transfected with NLRP3-Myc and treated with or without AP for 2 h.The interaction between NLRP3 and NEK7 was analyzed by immunoprecipitation and immunoblot.(b) The ATPase activity of NLRP3 with or without AP was measured by luminescence using the ADP-Glo™ Max assay.(c) LPS-primed THP-1 cells were treated with AP for 2 h and activated with nigericin (10 μM) for 30 min.The cell pellet was cross-linked by DSS (2.5 mM) for 30 min.ASC oligomerization was analyzed by immunoblot.(d,e) LPS-primed J774A.1 cells treated with AP for 2 h and activated with nigericin (10 μM) for 30 min.Representative immunofluorescence images of ASC speck formation (indicated by arrows) were taken by a confocal laser-scanning microscope (Carl Zeiss, LSM710, scale bar, 20 μm) (d).Graph representing the number of cells containing ASC specks in all treatments (e).

Figure 5 .
Figure 5. AP alleviates inflammatory response induced by LPS treatment in zebrafish.(a) Representative images of posterior areas of Sudan Black B-stained zebrafish embryos after LPS induction and AP treatment at 3 dpf.The upper panel shows the control group with DMSO (0.025%) treatment, the middle panel shows LPS inflammation group, and the bottom panel shows the LPS inflammation group after treatment with 10 µg/ mL AP.(b) Quantification of neutrophils in the posterior region of zebrafish after LPS induction and AP treatment.(c) Representative images of the caudal hematopoietic tissues after LPS induction and AP treatment at 3 dpf.Whole-mount in situ hybridization was conducted using an mpx probe.(d) Representative images of anterior Sudan Black B-stained zebrafish embryos following direct LPS injection and AP treatment at 3 dpf.(e) Quantification of the number of neutrophils in the anterior zebrafish embryos after LPS induction and AP treatment.All graphs represent the mean ± S.E.M. of individual values.P-values were calculated using an unpaired two-tailed Student's t-test.*P < 0.05; **P < 0.01; ***P < 0.001.